Neutralization activity of sera/IgG preparations from fully BNT162b2 vaccinated individuals against SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Kappa variants

In the present prospective study, 225 individuals in Kumamoto General Hospital, Japan, who received two-doses of BNT162b2 vaccine were enrolled/followed up over 150 days and neutralizing activity (NT50) of their sera and antiviral activity (EC50) of IgG purified from sera on day-60 post-1st-dose were determined against wild-type SARS-CoV-2 (SARS-CoV-2Wuhan) (n = 211) and 9 variants (Alpha, Beta, Gamma, Delta, and Kappa) (n = 45). Time-dependent changes of IgG-activity (n = 25) against SARS-CoV-2Wuhan and variants were also examined. Day-60 sera showed reduced NT50 by more than 50% against all variants examined, and greatest reduction was seen with Beta. IgG fractions of high-responders and moderate-responders showed similar fold-changes in EC50 against each variant compared to SARS-CoV-2Wuhan. Evaluation of EC50 of IgG obtained at different time-points (day-28 to -150) revealed time-dependent reduction of activity against all variants. However, against Delta, relatively long-lasting favorable antiviral activity (at least 150 days) was observed. Our data strongly suggest that the successful antecedent scale-up of mRNA-based vaccine administrations in Japan was the primary contributor to the lessening of the otherwise more devastating SARS-CoV-2 pandemic wave caused by the Delta variant. The present data that the effectiveness of vaccine against the then-dominant SARS-CoV-2 variant was likely associated with the moderation of the COVID-19 pandemic wave suggest that as in the case of influenza vaccines, the development of multivalent mRNA-based vaccines represent a generalizable approach to pre-emptively respond pandemic with mutable pathogens.

www.nature.com/scientificreports/ viral vector vaccines have also been used in certain countries 5,[7][8][9] . For instance, the adenovirus vector-based vaccine, AZD1222 (ChAdOx1 nCoV-19, Oxford/AstraZeneca), has reportedly showed 62.1% efficacy in initial clinical trials 10 . More recent study reported that robust immune responses accompanied by acceptable reactogenicity after heterologous vaccination of AZD1222 prime and BNT162b2 or mRNA-1273 boost 11,12 . Another adenovirus-based vaccine, Ad26.COV2.S (Janssen), has shown 85.4% prevention efficacy against severe/critical COVID-19 13,14 . However, the recent emergence of various SARS-CoV-2 variants with mutations in the spike-encoding region is raising global concerns about the efficacy of vaccines against such variants. The D614G and B.1.1.7 (Alpha/ N501Y) variants might have improved viral fitness, but apparently not immune escape 15,16 . However, the B.1.351 (Beta) variant, containing E484K substitutions, is reportedly represents a neutralization escape variant to convalescent sera 17 . A phase 3 trial of NVX-CoV2373 (Novavax), a recombinant protein-based nanoparticle vaccine indicated 86.3% efficacy to the more transmissible B1.1.7 (Alpha) variant in a post hoc analysis 18,19 . However, a phase 2b trial in South Africa showed 50.1% vaccine efficacy against the B1.351 (Beta) variant in a post hoc analysis 20 , suggesting that the Beta variant is less susceptible to antibodies elicited with the original Wuhan strain's antigens, which is in the composition of all the vaccines currently being evaluated 9 . P.1 (Gamma) variants also reported to evade antibody responses induced by previous infection or vaccination 21 .
Another recent concern is the emergence of a B.1.617.2 (Delta) variant, which was first detected in India, has been spreading around the world. This variant of concern (VOC) has reportedly less susceptibility to vaccineelicited protection and increased transmissibility beyond Alpha strains 22 . B1.617.1 (Kappa) variant, which belongs to the same lineage with the Delta variant and WHO classified as variant under monitoring (VUM), has characteristic E484Q mutation along with L452R and D614G mutations in the spike-encoding region, reportedly have reduced susceptibility to neutralization activity of therapeutic monoclonal antibodies and convalescent/ vaccinated sera, in experiments using pseudoviruses 23 .
In the present study, we examined neutralizing activity of sera obtained at day-60 post 1st dose from BNT162b2-vaccinated health care workers in Japan, against wild-type Wuhan strain (n = 211) and 9 distinct SARS-CoV-2 variants, including various VOCs (Alpha, Beta, Gamma, and Delta) and VUM (Kappa) (n = 45). All the SARS-CoV-2 strain/variants used in this study were infectious viruses, neither recombinant-virus nor pseudo-virus, and isolated from individuals at airport quarantine station or hospital in Japan. We also investigated antiviral activity of IgG obtained from day-60 sera of 45 participants (classified as group of high responders and group of moderate responders, by the results of neutralizing activity of each serum against wild-type Wuhan strain) against wild-type and various variants. Finally, we eluted IgG fractions from sera of 25 participants at day-28, -90, and -150, then examined time-dependent alternations of antiviral activity of each IgG against Wuhan strain, VOCs, and VUM.

Results
Immune response in the participants on day-60 post 1st dose. We obtained blood samples for neutralizing activity evaluation from a total of 225, 220, 211, 210, and 208 vaccine recipients on days 7, 28, 60, 90, and 150 post 1st doses, respectively 24 . Demographic characteristics of the participants are shown in Table 1. As of the time of enrollment, the average age of the participants was 41.8 years (range 21 to 72 years), and 69.8% of the participants were female serving as a physician, nurse, paramedical staff, or administrative staff. None of the participants was in the immunodeficient state or was receiving immunosuppressants or steroids. Table 1. Demographic characteristics of the participants. *None of the participants were in immunodeficient states or were receiving immunosuppressants or steroids. Two participants had previously experienced SARS-CoV-2 infection and were excluded from the present study since day-28. None of other participants experienced COVID-19-related symptoms and proved to be negative for antibodies against serum SARS-CoV-2 nucleocapsid throughout the study period. In the present study, we focused upon the blood samples taken on day-60 post 1st dose from 211 vaccinated participants. First, we determined the neutralizing activity of each serum against cytopathic effect induced by wild-type SARS-CoV-2 05-2N infection and replication in the VeroE6 cell-based assay. As shown in Fig. 1, 50% neutralization titer (NT 50 ) levels were substantially diverse among the participants: the average NT 50 value was 284 (range 30-1290), and five participant's sera showed NT 50 values less than cut-off level (< 20).
High and moderate responder's sera show reduced anti-SARS-CoV-2 activity against various VOCs and VUM. Next, we selected and designated day-60 participants, whose sera had NT 50 values against SARS-CoV-2 05-2N greater than 500 as high responders (n = 15; shown in red), and participants, whose sera had NT 50 values around average of all participants (range 220 to 380) as moderate responders (n = 30; shown in blue) ( Fig. 1), and evaluated neutralizing activity of each serum belonging above mentioned two groups against nine infectious SARS-CoV-2 variants; variants of concerns (VOCs) and variants under monitoring (VUM), including Alpha, Beta, Gamma, Delta, Kappa, and E484K/D614G-amino acid subs carrying variants (E484K variants).
Overall, sera in both groups showed reduced neutralizing activity by greater than 50% against all variants examined in this study compared to those against wild-type SARS-CoV-2 05-2N , and the greatest reduction of neutralizing activity occurred with the Beta variant.
IgG fractions obtained from high and moderate responders' sera show different properties regarding anti-SARS-CoV-2 activity against various VOCs and VUM. In attempt to further quantify anti-SARS-CoV-2 activity induced by BNT162b2, we obtained IgG fractions from each of high and moderate responder's serum and carried out the VeroE6 TMPRSS2 cell-based anti-SARS-CoV-2 assay determining EC 50 Figure 1. Neutralization of wild-type SARS-CoV-2 05-2N by day-60 sera. Neutralization activity of day-60 sera obtained from participant were shown. Day-60 participants whose sera showed NT 50 values against SARS-CoV-2 05-2N greater than 500-fold were classified as high responders (open red circles, n = 15), and participants whose sera showed NT 50 values around average of all participants (range 220 to 380-fold) were categorized as moderate responders (open blue circles, n = 30). www.nature.com/scientificreports/ (50% effective concentration: μg/ml) value of each IgG fraction against wild-type SARS-CoV-2 05-2N and nine distinct SARS-CoV-2 variants. We believe that such attempt can exclude any effects caused by non-IgG factors existing in sera, enable to evaluate actual neutralizing activity of IgG alone as induced by the BNT162b2 vaccination against SARS-CoV-2.

Strains
We also carried out comparison between NT 50 values (sera) and EC 50 values (IgG preparations) of randomly selected participants [the high (n = 15) and moderate (n = 30) responders: a total of 45 participants] using data obtained various strains. As shown in Fig. S1, significant inverse correlations between the two values against the ancestral Wuhan strain and 4 VOCs (Alpha, Beta, Gamma, and Delta variants) were observed (Spearman ρ values = − 0.82 to − 0.59; p < 0.0001).

Evaluation of anti-SARS-CoV-2 activity of IgG fractions obtained at different time points reveals relatively long-lasting antiviral activity of IgG against Delta-variant. Next, we also eluted
IgG fractions from sera of 25 participants at day-28 and day-90 (n = 13 for IgG high s and n = 12 for IgG moderate s), then determined EC 50 values of each IgG fraction against SARS-CoV-2 05-2N , various VOCs, and VUM.
As shown in Fig. 4 and Table 4, both IgG groups maintained good anti-SARS-CoV-2 activity against SARS-CoV-2 05-2N , average EC 50 values of IgG high s and IgG moderate s from day-28 sera were 4.3 and 6.9 μg/ml, respectively, and 13.7 and 36.2 μg/ml, respectively, for the IgG eluted from day-90 sera.
IgG from day-28 sera of both groups showed reduced anti-SARS-CoV-2 activity against Beta variant, average EC 50 values of IgG high s and IgG moderate s were 60.8 and 90.8 μg/ml, respectively, and values continued to increase up to 92.6 and 189.3 μg/ml, respectively, for IgG from day-90 sera (Fig. 4B, Table 4A).
Against Gamma variant, IgG from day-28 sera of both groups relatively maintained anti-SARS-CoV-2 activity with EC 50 values lower than 50 μg/ml (30.3 μg/ml for IgG high s and 48.2 μg/ml for IgG moderate s), although values elevated up to 57.4 and 124.6 μg/ml for day-90 IgG high s and IgG moderate s, respectively (Fig. 4B, Table 4A).
As for Delta variant, both IgG groups from day-28 sera showed good anti-SARS-CoV-2 activity, EC 50 were 10.3 μg/ml for IgG high s and 31.8 μg/ml for IgG moderate s, and maintained relatively favorable EC 50 values for IgGs from day-90 sera, EC 50 were 22.0 μg/ml for IgG high s and 65.9 μg/ml for IgG moderate s (Fig. 5, Table 4B). This favorable antiviral activity of IgG high s against Delta variant was maintained at least 150 days post 1st dose (Fig. 5, Table 4B).
Against Kappa variant, IgG from day-28 sera relatively maintained anti-SARS-CoV-2 activity with EC 50 values of 20.1 μg/ml for IgG high s and 51.0 μg/ml for IgG moderate s, and EC 50 values elevated up to 62.1 and 124.1 μg/ml for day-90 IgG high s and IgG moderate s, respectively (Fig. 4C, Table 4A). As for E484K variant, similar results were observed with those of Delta variant (Fig. 4C, Table 4A).

Discussion
In our previous report, we examined neutralizing activity and spike S1-binding antibody (IgG and IgM) response in 225 healthy individuals [physicians (n = 36), nurses (n = 125), and other healthcare professionals (n = 64)], who received two doses of 30 µg BNT162b2 (Pfizer/BioNTech with 3-week interval between 1st and 2nd doses) vaccine in February 2021 in Japan, and investigated the correlation among neutralizing activity levels, S1-binding-IgG and -IgM levels, genders, and adverse events, and time-dependent decline of BNT162b2-elicited immune response 24 . In the present study, we focused on the blood samples collected at 60 days post 1st dose of BNT162b2 vaccination and determined neutralization activity represented by 50% neutralization titers (NT 50 ; fold) of each serum and antiviral activity represented by 50% effective concentrations (EC 50 ; μg/ml) of each purified IgG fraction by employing the inhibition of virus-induced cytopathic effect employing the target VeroE6 TMPRSS2 cells and infectious SARS-CoV-2s. Among infectious SARS-CoV-2 strains we used, a Beta variant, containing K417N, E484K, N501Y, and D614G mutations in the spike-encoding region, showed the most significantly reduced susceptibility profile with % reductions of average NT 50 being 88.5 and 83.4% for high and moderate responders, respectively, compared to those against SARS-CoV-2 05-2N . Liu et al. previously reported that plaque reduction neutralization assay employing blood samples from 15 participants on 2-4 weeks post 2nd dose of BNT162b2 and recombinant virus that contained all the spike mutations of B.1.351 (Beta), showed 63.5% reduction in 50% plaque reduction neutralization titer (PRNT 50 ) compared to that against a parental USA-WA1/2020 strain 25    www.nature.com/scientificreports/ interpretable because there are some differences between their and our experiments, such as assay methods, origins of virus used (recombinant virus or clinically isolated virus), numbers of selected participants, and the timing of blood collection. The basic design of the present prospective study was (i) to screen the entire cohort (n = 225) in a middle-sized hospital in Japan, where 225 healthcare professionals voluntarily participated in the current study for determining the neutralizing activity of their sera/IgG preparations using the infectious SARS-CoV-2 (the ancestral Wuhan strain)-employed, cell-based assay system; and (ii) to examine the sera/IgG preparations' neutralizing activity against various SARS-CoV-2 variants of randomly selected high (n = 15) and moderate (n = 30) responders from the same cohort. The scheme was that as many as 45 randomly selected participants' sera/IgG preparations will produce a set of data, which represents the nature of immunologic features of the entire cohort. Thus, throughout the present study, the sera/IgG preparations from the 45 participants were examined for their neutralizing activity against various variants of concerns (VOCs; Alpha, Beta, Gamma, and Delta variants) and a variant under monitoring (VUM; Kappa variant).
When the decline of neutralization activity is compared on day-28, -60, -90, and -150 post-1st dose, neutralization of high responders' sera tends to be slow and the activity remained effective longer than that of moderate responders' sera. This is perhaps due to the MOI effects in the cell-based analysis where if the blockade of viral infectivity and replication is not less, more daughter virions would be produced, thus increasing greater MOI effects than when the blockade is more potent. It is assumed that similar events should occur in the in vivo Table 4. Anti-SARS-CoV-2 activity of IgG fractions eluted from day-28, 60, 90, and 150 sera of high and moderate responders against VOCs and VUM. Average EC 50 values of IgG fraction eluted from sera of high (n = 13) or moderate (n = 12) responders, taken on day-28, -60, -90, and -150 post 1st vaccine doses, are indicated. *Fold change of EC 50 values compared to that of day-28 IgG. **Mean of fold change was calculated by arithmetic mean of each participant's fold change at day-60, -90, and -150 against day-28. The difference between high and moderate responders using Student's t-test.  , and SARS-CoV-2 TY7-501 (Gamma; Japan/Brazil January 2021) (https:// www. who. int/ en/ activ ities/ track ing-SARS-CoV-2-varia nts/). However, the susceptibility of such variants to neutralization activity of the sera/IgG preparations did not get less in that temporal order. Notably, among the variants examined in the present study, the Beta variant (TY8-612) was least susceptible to the neutralizing activity of IgG preparations from both high and moderate responders, while the Alpha and Delta variants, which appeared later than the Beta variant, were more susceptible to neutralizing activity than the Beta variant (Figs. 2, 3). These data probably suggest a co-expansion nature of each SARS-CoV-2 variant among neighboring and even distant countries and regions with diverse sources and through diverse transmission routes. Moreover, the chronological order of the predominant emergence of the variants is influenced by the transmissibility and pathogenicity acquired by each variant. Thus, it is not surprising that the order of susceptibility of variants to neutralization of sera/IgG preparations from the vaccinated individuals did not match the temporal order of the emergence of the variants examined in the current study.
When we examined and analyzed the time-dependent reduction (day-28 to -90 post 1st vaccine dose) in IgG's antiviral activity against all the SARS-CoV-2s used in the present study (Figs. 4, 5, Table 4), two variants originated from India (Delta and Kappa variants) showed distinguish profiles against neutralizing activity elicited by BNT162b2 vaccination. The Kappa variant showed moderate to high immune evasion profile probably due to characteristic E484Q mutation in the spike-encoding region, but mutation in this amino-acid does not exist in the Delta variant. It was interesting finding in the present study that purified IgG from sera obtained from 2 doses of BNT162b2 vaccinated Japanese individuals showed relatively long-lasting (at least 150 days post 1st vaccine dose) favorable neutralizing activity against the Delta variant (Fig. 5, Table 4). In Japan, the most severe SARS-CoV-2 pandemic (5th wave) arose from end of July-2021 26 (Fig. 6), concurrently with an opening of the Olympic games, and % of COVID-19 by Delta variant infection-confirmed cases reached ~ 90% of 5th wave in early-August. The Japanese government has been accelerating SARS-CoV-2 vaccination recently (since mid-June 2021), number of vaccine administrated doses in Japan reached over 100 million doses on August-10th, and since 3 weeks later, number of newly confirmed SARS-CoV-2 positive cases began to decline rapidly afterwards 26,27 (Fig. 6). These data and our results suggest that the successful antecedent scale-up of mRNA-based vaccination in Japan since June-2021 probably was the primary contributor to the moderation of the otherwise devastating SARS-CoV-2 pandemic, which was caused by the Delta variant susceptible to BNT162b2-elicited immunity. Indeed, the SARS-CoV-2 vaccines used in Japan by Aug-10th, 2021 were mRNA-based vaccines only; BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna). In addition, the percentages of total vaccine doses of BNT162b2 and mRNA-1273 used in Japan by Aug-10th, 2021 were 81% and 19%, respectively. Thus, as for the possibility that www.nature.com/scientificreports/ natural-infection-acquired-immunity had moderated the 5th wave, the percentage of total SARS-CoV-2-infected cases in Japan at the beginning of the pandemic (Jan-22nd, 2020 to Aug-10th, 2021) was less than 1% of the entire Japanese population (0.83%); hence, it's unlikely that natural-infection-acquired-immunity contributed to the moderation of the 5th wave of SARS-CoV-2 infection in Japan. The present data that the effectiveness of vaccine against the then-dominant SARS-CoV-2 variant was likely associated with the moderation of the COVID-19 pandemic wave suggest that as in the case of influenza vaccines 28,29 , the development of multivalent (multi-SARS-CoV-2-spike-antigen in the case of COVID-19) mRNA-based vaccines represent a generalizable approach to pre-emptively respond pandemic with mutable pathogens.

Materials and methods
Serum specimens. Serum samples were collected from 225 vaccinated health care workers at JCHO Kumamoto General Hospital (Kumamoto, Japan). Samples were analyzed at Kumamoto University in Kumamoto and the National Center for Global Health and Medicine (NCGM) in Tokyo. The Ethics Committee from the Kumamoto General Hospital and NCGM approved this study (Kumamoto General Hospital No. 180, and NCGM-G-004176-00). Each participant provided a written informed consent, and this study abided by the Declaration of Helsinki principles. The vaccination (on days 0 and 21, 30 μg/each dose) and serum collection (from day-7, -28, -60, -90 and -150 post 1st dose; number of participants on day-7, -28, -60, -90, and -150 were 225, 220, 211, 210, and 208, respectively) were conducted and carried out.